Direct measurement of single-channel Ca2+ currents in bullfrog hair cells reveals two distinct channel subtypes

To confer their acute sensitivity to mechanical stimuli, hair cells employ Ca 2+ ions to mediate sharp electrical tuning and neurotransmitter release. We examined the diversity and properties of voltage-gated Ca 2+ channels in bullfrog saccular hair cells by means of perforated and cell-attached patch-clamp techniques. Whole-cell Ca 2+ current records provided hints that hair cells express L-type as well as dihydropyridine-insensitive Ca 2+ currents. Single Ca 2+ channel records confirmed the presence of L-type channels, and a distinct Ca 2+ channel, which has sensitivity towards ω-conotoxin GVIA. Despite its sensitivity towards ω-conotoxin GVIA, the non-L-type channel cannot necessarily be considered as an N-type channel because of its distinct voltage-dependent gating properties. Using 65 m m Ca 2+ as the charge carrier, the L-type channels were recruited at about –40 mV and showed a single-channel conductance of 13 pS. Under similar recording conditions, the non-L-type channels were activated at ∼–60 mV and had a single-channel conductance of ∼16 pS. The non-L-type channel exhibited at least two fast open time constants (τ o = 0.2 and 5 ms). In contrast, the L-type channels showed long openings (τ o =∼23 ms) that were enhanced by Bay K 8644, in addition to the brief openings (τ o = 0.3 and 10 ms). The number of functional channels observed in patches of similar sizes suggests that Ca 2+ channels are expressed singly, in low-density clusters (2–15 channels) and in high-density clusters (20–80 channels). Co-localization of the two channel subtypes was observed in patches containing low-density clusters, but was rare in patches containing high-density clusters. Finally, we confirmed the existence of two distinct Ca 2+ channel subtypes by using immunoblot and immunohistochemical techniques.